Compound for inhibiting the influx of polymorphonuclear leukocytes (PMNS) in a tissue, its selection, pharmaceutical compositions and use

ABSTRACT

The invention relates to a compound suitable for inhibiting the influx of polymorphonuclear leukocytes (PMNs) into a tissue involved in a chronic inflammatory disease. The compound according to the invention is capable of forming a complex with N-acetyl-Pro-Gly-Pro. The invention also relates to a method of selecting such a compound, a pharmaceutical composition and an application of the compound.

[0001] Compound for inhibiting the influx of polymorphonuclearleukocytes (PMNs) in a tissue, method of selecting such a compound, apharmaceutical composition, and an application of the compound

[0002] The present invention relates to a compound suitable forinhibiting the influx of polymorphonuclear leukocytes (PMNs) into atissue involved in a chronic inflammatory disease in a mammal, whichcompound, in the presence of equimolar amounts of a)N-acetyl-Pro-Gly-Pro (“N-AcPGP”), b) a compound designated as 4RTR withthe formula I

[0003] which is capable of forming a complex with N-AcPGP, and c) thecompound, inhibits complex formation between N-AcPGP and 4RTR by atleast 10% due to N-AcPGP being bound by the compound.

[0004] As the person skilled in the art knows, in the formula 4RTR, R,T, G, K and A stand for arginine, threonine, glycine, lysine, andalanine. Influx of polymorphonuclear leukocytes (PMNs), such asneutrophilic and eosinophilic granulocytes, plays a role with chronicinflammatory diseases. In the present application chronic inflammatorydiseases are understood to include all inflammatory diseases induced byprolonged exposure to a foreign substance or where no exposure to aforeign substance is involved. These include in particular inflammatorydisorders of the lungs, joints, and bowels, among which autoimmunediseases, (severe) asthma and lung emphysema. With such chronicinflammatory diseases of this kind, PMNs migrate from peripheral bloodinto a tissue where they cause damage to said tissue. Normally, theinflux of PMNs should occur only to combat an infection with potentiallyharmful microorganisms. The influx of PMNs causes, due to the secretionof additional signalling substances such as leukotrienes, an influx ofother inflammatory cells, thus increasing the damage to the tissue. Acompound binding N-AcPGP, a signal substance activating PMNs,significantly inhibits the pre-activation of PMNs and consequently theinflux of the neutrophilic granulocytes. The phrase “as a result ofN-AcPGP being bound by the compound” means that the compound is capableof forming a complex with N-AcPGP but not with 4RTR. Using well-knowntechniques, such as a microcaloric technique, the person skilled in theart is quite well able to establish that such a complex has been formed(Ref. 1 and 2).

[0005] Preferably, the compound inhibits the formation of the complex byat least a 25%, preferably by at least 50%, more preferably by at least70%, and most preferably by at least 90%.

[0006] Such compounds are more effective and, as a smaller quantity isrequired for a treatment, they are potentially also more interestingfrom an economic point of view.

[0007] According to the first embodiment the compound is a peptide.

[0008] The use of a peptide or a compound comprising a peptide, such asRTR or its oligomers, increases the chance of biological compatibility,as its decomposition products comprise natural amino acids.

[0009] If it is desirable that the peptides stay active in the bloodlonger, the peptide may comprise one or more D-amino acids. According toan alternative embodiment, the compound is a peptidomimetic in which theside chain is located on the nitrogen atom.

[0010] Suitable compounds comprise at least two XTR units which areinterconnected by means of one or more spacer units. X is any arbitraryamino acid and suitably a natural amino acid such as arginine. Suitably,spacer units each have a length (from RTR unit to RTR unit) of 2 to 10atoms. To make a spacer it is convenient to use one or more glycinemolecules, or synthetic amino acids such as 3-aminopropionic acid. Asuitable compound is preferably a branched compound, in which at leastthree branches comprise an RTR chain. To achieve branching, the compoundsuitably comprises natural or synthetic di-amino acids (lysine,di-aminoproprionic acid, di-aminobutyric acid). Usually each compoundwill comprise at the most 100 RTR units, suitably 4 to 6 units. Asposited above, instead of the natural amino acids of RTR it is alsopossible to use the D-variants or variants in which the side chain islocated on the nitrogen atom of the backbone. Preferably, a PGP-bindingunit, and in particular the compound itself, has a molecular weight ofless than 5000, such as <4000.

[0011] The invention also relates to a method of screening a number ofcompounds with respect to their capability to inhibit the formation of acomplex between 4RTR and N-AcPGP, screening occurring by means of anassay comprising a competition reaction between i) a compound A to beassayed and ii) a compound B capable of binding N-AcPGP having anequilibrium constant K of 5.10⁻⁵ M¹ or lower, compound A and compound Bbeing in competition for the binding to a Pro-Gly-Pro (PGP)-comprisingcompound C, with which the compound B is able to complex, having anequilibrium constant K of 5.10⁻⁵ M⁻¹ or lower, and wherein a thusassayed compound A is selected having an equilibrium constant K for thereaction between compound A and the PGP-comprising compound C of 10⁻⁴M⁻¹ or lower.

[0012] Such a method makes it possible to select compounds, that can beassayed for further pharmacological properties such as in blood halflife, toxicity and the like, as is well-known to the person skilled inthe art of pharmacology.

[0013] Preferably, a compound A is selected having an equilibriumconstant K of 10⁻⁵ M⁻¹ or below, preferably 10⁻⁶ M⁻¹ or below, morepreferably 10⁻⁷ M⁻¹ or below, and most preferably 10⁻⁸ M⁻¹ or below.

[0014] Such high affinity compounds A can be used to great advantage forthe preparation of a pharmaceutical composition, as will be describedbelow.

[0015] Suitably the compound C is N-R-Pro-Gly-Pro (“N-R-PGP”), wherein Ris branched or linear alkyl- or acyl group having 1 to 6 carbon atoms,preferably 1 to 2 carbon atoms. A suitable compound C is N-Ac-PGP orN-methyl-Pro-Gly-Pro.

[0016] Such PGP-comprising compounds are simple to synthesize and beused for the selection of suitable compounds A. As the compound B forbinding the PGP-comprising compound C it is possible to use, forexample, 4RTR.

[0017] Preferably the assay is a homogenous assay, such as one based onfluorescence (de)polarization or internal energy transfer.

[0018] This is a quick and efficient manner of screening a considerablenumber of compounds and selecting an inhibiting compound.

[0019] The invention also relates to compounds suitable for inhibitingthe influx of PMNs in a tissue involved in a chronic inflammatorydisease in a mammal, which compound E, in the presence of an equimolaramount of N-acetyl-Pro-Gly-Pro (“N-AcPGP”), competes for binding to aPMN and inhibits the binding of N-AcPGP to the PMN by at least 10% as aresult of N-AcPGP being bound by the compound, and which compound E doesnot induce activation of the PMN.

[0020] A compound E which does not engage a signal compound such asN-acetyl-Pro-Gly-Pro or N-methyl-Pro-Gly-Pro but its receptor on aneutrophilic granulocyte, is also suitable for inhibiting the influx ofPMNs.

[0021] Suitably the compound E inhibits binding by at least 25%,preferably by at least 50%, more preferably by at least 70% and mostpreferably by at least 90%. Again, the compound may be a peptide orpeptidomimetic. In that case the above-described advantages concurrentlyapply.

[0022] According to an alternative embodiment the compound is anantibody, preferably a monoclonal antibody, and most preferably a humanor humanized monoclonal antibody, or fragments thereof.

[0023] With the aid of such antibodies or fragments thereof, it ispossible to effectively trap the PGP-comprising signal substance. Forlong-term activity it is preferred that the antibody be adapted to thetype of mammal. For humans, therefore, use is made of human antibodiesor of humanized antibodies made by using techniques that are well knownin the art.

[0024] The invention also relates to a method of screening a number ofcompounds with respect to their ability to inhibit the binding ofN-AcPGP to a PMN, wherein screening occurs

[0025] a) by using an assay comprising a competition reaction between acompound E to be assayed and a compound D for the binding to a PMN,wherein compound D is capable of competing with N-acetyl-Pro-Gly-Pro(“N-AcPGP”) for binding to a PMN and at equimolar concentrations of thecompound D and N-AcPGP, inhibits the binding of N-AcPGP by at least 50%,and at equimolar concentrations of the compound E and compound D,inhibits the binding of D to the PMN by at least 10%, and

[0026] b) by contacting the compound E with a PMN and selecting acompound E which substantially does not induce activation of the PMN.

[0027] This is an efficient manner of selecting compounds E whichinhibit the binding of the PGP-comprising signal substance to itsreceptor on the PMN. In practice it will be preferred to first carry outstep a) and then step b).

[0028] Suitably compound D is N-R-Pro-Gly-Pro, wherein R is a branchedor linear alkyl or acyl group having 1 to 6 carbon atoms, preferably 1to 2 carbon atoms, such as N-Ac-PGP or N-methyl-PGP.

[0029] According to a suitable embodiment the assay in respect to theactivation of the PMN comprises measuring the polarization of the PMN.

[0030] The polarization can be established in a simple manner, such asdescribed by Haston, W. S. and Shields, J. M. “Neutrophil Leukocytechemotaxis: a simplified assay for measuring polarizing responses tochemotactic factors”. J. Immunol. Methods. 81: 229-237 (1985).

[0031] According to a suitable embodiment the inhibition of the bindingis established with the aid of flow cytometry.

[0032] The inhibition of the binding, and also the activation of the PMNcan be established in a simple manner with the aid of flow cytometry.

[0033] The invention further relates to a pharmaceutical compositioncomprising a compound according to the invention or a compound selectedwith a method according to the invention, as well as a pharmaceuticallyacceptable carrier or excipient.

[0034] The invention also relates to an application of a compoundaccording to the invention, or a compound selected with a methodaccording to the invention, for the preparation of the pharmaceuticalcomposition suitable for the treatment of a chronic inflammatory diseasein a mammal, such as a disease belonging to the group comprising chronicinflammatory bowel diseases, rheumatoid arthritis, and other auto-immunediseases, heart diseases that are characterized by an influx ofneutrophilic granulocytes, such as heart ischemia, Adult RespiratoryDistress Syndrome (ARDS), asthma and lung emphysema.

[0035] The invention will now be explained in more detail with referenceto the following examples and with reference to the drawing in which

[0036]FIG. 1 is a histogram showing the effect of 4-RTR on thedilatation of bronchial tubes; and

[0037]FIG. 2 is a histogram showing the effect of 4-RTR in a model ofInflammatory Bowel Disease.

[0038] Peptide Synthesis

[0039] Peptide synthesis is well established in the art. Peptides mayalso be obtained commercially as a custom-made product. The followingdescription of the synthesis of 4RTR and N-AcPGP are just respectiveroutes to obtain said compounds.

[0040] Preparation of 4RTR

[0041] The sequence RTR was chosen using a method described by Blalock(Nature Medicine, 1, p. 876 (1995)). The two glycine residues in 4RTRserve as a spacer. The number of glycine residues may vary (for example,one glycine residue instead of two also gives excellent results) and,indeed, spacers other than glycine may be used. Amino acids may be L or,to have a greater stability against (proteolytic) degradation in vivo,D. The D configuration does not appear to have a significant effect onits ability to bind N-acetylPGP (results not shown).

[0042] The RTR tetrameric peptide((H₂N-Arg-Thr-Arg-Gly-Gly)₂-Lys)₂-Lys-Ala-CONH₂), containinglaevorotatory (L) RTR sequences, was synthesized using Solid PhasePeptide synthesis following Fmoc methodology on a 9050 Peptidesynthesizer from Perceptive Biosystem. This (L)-RTR tetrameric peptidewas synthesized starting from a Fmoc-Alanine-PEG-PS resin, with eitherone or two coupling cycles with Fmoc-K-moc-OH activated with HATU/DIPEA.The following couples were achieved using Fmoc amino acids activatedwith HATU/DIPEA. The Fmoc deprotection reagent was 1% DBU, 1% Piperidinein dimethylformamide. The peptide was cleaved from the resin by adding10 ml of trifluoroacetic acid (TFA)/phenol/thioanisol/H₂O/ethanedithiol93/2/2/2/1 and incubated at room temperature from 5 hours. The mixturewas filtered and the peptide precipitated in cold ethyl ether. Theprecipitate was collected and solubilized in H₂O for lyophilization. Thepeptide was purified by reverse phase high performance liquidchromatography (RP-HPLC), using a Dynamax RP C18 (300×10 mm i.d.), andequilibrated at 3 ml/min using a linear gradient from 5% CH₃CN to 60%CH₃CN in 0.1% TFA in 40 minutes. The fractions containing the peptidewere acidified with 1 N HCl to help in the elimination of TFA, andlyophilized. Peptide identity was confirmed by time of flight matrixassisted laser desorption ionization mass spectroscopy. Purity wasconfirmed by analytical RP-HPLC.

[0043] The RTR tetrameric peptide((H₂N-d-Arg-d-Thr-d-Arg-Gly-Gly)₂-Lys)₂-Lys-Ala-CONH₂), containingdextrorotatory (D) RTR sequences (only RTR was d confirmation), wassynthesized manually starting with 3 g of the Fmoc-Pal-Peg-PS resin withan initial substitution of 0.2 mmol/g of resin. DMF was used as solventfor the coupling steps and the washing steps, while Fmoc was achievedwith 1% DBU/2% Piperidine in DMF. Monitoring of the coupling anddeprotection steps was conducted using the Kaiser assay. All amino acidswere doubly coupled for one hour, using as activating reagents, HOAt forthe O-Pentafluorophenyl ester amino acid and HATU/DIPEA for the freeacids. An excess of 5 equivalents of amino acid over the resinsubstitution was used for alanine and the first lysine, 10 equivalentsfor the second lysine, and 20 equivalents for the following amino acids.The (D)-RTR tetrameric peptide was cleaved from the resins and purifiedas for the (L)-RTR peptide.

[0044] Preparation of N-AcPGP

[0045] The dipeptide t-Boc-PG was coupled to Pro-Merrifield resin usingthe dicyclohexylcarbodiimide/1-hydroxybenzotriazole procedure. After theremoval of the N-terminal protection and acetylation using aceticanhydride, the peptide was cleaved from the resin using anhydroushydrofluoric acid. The product was purified on a silica gel column usingchloroform:methanol 90:10 v/v) as the eluent. Homogeneity was confirmedby RP-HPLC on a Vydac C18-analytical column equilibrated at a flow rateof 1.2 ml/min and eluted with a linear gradient from 0% to 30%acetonitrile in water (0.1% trifluoroacetic acid) in 30 minutes. Peptideidentity was confirmed by Electrospray Mass Spectrometry(Perkin-Elmer-Sciex API-3). Quantitative amino acid analysis wasperformed to show the correct ratio of amino acids and to determine thepeptide content for calculation of the final concentration.

EXAMPLE 1

[0046] Inhibition of Neutrophilia in the Mouse, Induced byPGP-Comprising Compounds

[0047] Solutions were prepared of N-Ac-PGP (0.36 mg/ml; 10 ⁻³ M) andN-methyl-PGP (0.32 mg/ml; 10⁻³ M) in phosphate-buffered physiologicalsalt (PBS). In an atomizing chamber, 6-8 weeks old Balb/c mice (groupsof 4 mice) were subjected for 30 minutes to either N-acetyl-PGP orN-methyl-PGP. Control mice only received PBS. One hour prior tosubjection in the atomizing chamber and one hour after the start ofsubjection each mouse received 50 μl of 1 mg/ml 4RTR. The treatment isoutlined in the Table I below. TABLE I Outline of treatment: groupnumber pre-treatment atomization n = 4 PBS 4RTR PBS N-a-PGP N-m-PGP 1 +− + − − 2 + − − + − 3 + − − − + 4 − + + − − 5 − + − + − 6 − + − − +

[0048] 5 hours after the onset of subjection the mice were sacrificed byadministering an overdose of Nembutal. After the application of atracheal canula the lungs were washed with 4 ml PBS. The lung-washliquid was centrifuged (5 min.; 4° C.; 580 g) and the cell pellet wastaken up in 150 μl PBS. The cells were counted under the microscope. Todistinguish between neutrophilic and eosinophilic granulocytes andmononuclear cells (haematoxylin/eosin dye, H/E dye: Diff-Quick, Merz &Dade, A.G. Dübingen, Switzerland), the cytospin technique was applied(Buckley, T. L. and Nijkamp, F. P., “Airways hyperreactivity andcellular accumulation in a delayed-type hypersensitivity reaction in themouse: modulation by capsaicin-sensitive nerves”. Am. J. Respir. Crit.Care Med. 149: 400-407 (1994)).

[0049] Table II shows that the atomization of N-AcPGP and N-methyl-PGPresults in the influx of neutrophilic and eosinophilic granulocytes inthe lungs. Pretreatment with 4RTR resulted in a substantially totalinhibition of the influx of neutrophilic granulocytes. TABLE II Effectof treatment with 4RTR. The effect of 4RTR treatment on the PGP-inducedPMN influx in the mouse lung Treatment Total Neutros Eos Monos PBS/PBS30.4 ± 4.3 0.6 ± 0.3 0.0 ± 0.0 29.8 ± 4.2 PBS/N-AcPGP 49.5 ± 3.1 20.1 ±7.5* 1.1 ± 0.6 28.3 ± 8.6 PBS/N-methylPGP 46.1 ± 3.2 11.9 ± 3.6* 0.2 ±0.1 33.9 ± 3.4 4RTR/PBS 32.6 ± 1.7 0.5 ± 0.4 0.0 ± 0.0 32.1 ± 1.94RTR/N-AcPGP 26.3 ± 3.3 1.3 ± 0.4 0.2 ± 0.2 24.7 ± 3.2 4RTR/N-methylPGP25.9 ± 3.1 0.9 ± 0.3 0.0 ± 0.0 25.9 ± 3.1

EXAMPLE 2

[0050] Induction of Lung Emphysema by N-AcPGP and Inhibition by 4-RTR

[0051] On day 0, 2 and 7, two groups C75Bl/6J mice received 30 minutes'treatment with an aerosol (in an atomizing chamber as described inexample 1) comprising (i) vehicle (phosphate-buffered physiological saltsolution), (ii) N-AcPGP (0.4 mg/ml) or (iii) N-AcPGP (0.4 mg/ml) and4-RTR (1 mg/ml). 30 Minutes prior to the aerosol, the mice of group (i)and (ii) received intranasal vehicle and the mice of group (iii), 4RTR(1 mg/ml). 24 Hours after the final treatment the first group of mice(i.e. on day 8) exhibited a 2-fold increase of the total number ofinflammatory cells in the bronchial-alveolar washing liquid incomparison with the vehicle control (PBS). 4RTR produced approximately a75% inhibition of this increase. This 4RTR inhibition is specific forN-AcPGP, because 4RTR does not significantly inhibit the increase of thenumber of cells caused by the bacterial chemotactic peptide FMLP. (seeTable III). TABLE III 4RTR inhibits the increase of cells in thebronchial- alveolar liquid induced by N-AcPGP in C57B1/6J mice. (×10⁵)Vehicle N-AcPGP 4RTR + NacPGP 4RTR FMLP 4RTR + FMLP Total 30 ± 3  55 ±1* 29 ± 1  36 ± 8  61 ± 5*  58 ± 12* number infl. cells Number 0.3 ± 0.111.7 ± 0.8* 1.2 ± 0.6 0.1 ± 0.1 10.7 ± 3*   6.8 ± 1*  PMNs

[0052] On day 21 the mice of the second group (which on day 0, 2, and 7were treated in the same manner as the mice of the first group), weresacrificed by means of an overdose of anaesthetic (Nembutal) and thelungs were isolated and fixed by filling the lungs with Carnoy'sfixative under a liquid pressure of 25 cm. The lungs were prepared forhistological analysis. The mean linear intercept (Lm), indicative foralveolar dilatation, was calculated by placing a grid over theillustrations of 5 separate fields (300× magnification) per mouse lung.The total length of the lines was divided by the number of intercepts,resulting in the Lm, providing a measure for the average size of thebronchial volume of the lung.

[0053] Treatment with N-AcPGP resulted in a significant increase of theLm, which was prevented by simultaneous treatment with 4-RTR (seeFIG. 1. p<0.05 in comparison with the vehicle. ANOVA succeeded by theassay of Dunnett for multiple comparison). This result was inconcurrence with the change in the number of inflammatory cells.

[0054] Therefore these results show that 4-RTR has an inhibiting effecton the development of inflammation-related lung emphysema induced byN-AcPGP.

EXAMPLE 3

[0055] 4-RTR Treatment in a Model of Inflammatory Bowel Disease (IBD)Model

[0056] On day 0, the Balb/c mice were sensibilized withdinitrofluorobenzene (DNFB, 0.5% w./vol.), 50 μl being rubbed on theshorn belly and 50 μl on the top of the paws. On day 1 an additional 50μl DNFB (0.5% w./vol.) was rubbed on the belly of the mice.

[0057] On day 5 the mice were challenged with dinitrobenzene sulphonicacid (DNS, 1.2% w./vol., 25 μl rectal; 2 cm deep).

[0058] Minutes before and 30 minutes after the challenge the miceunderwent a rectal treatment (2 cm deep) with PBS or 4-RTR (5 pg/50 μl).

[0059] 24 Hours after the challenge the results were tallied on thebasis of the consistency of the feces, with a tally of 0, 1, or 2(0=normal, 2=diarrhoea and 1=in between).

[0060] The results were plotted (FIG. 3) as averages of the group (n=6).

[0061] 4-RTR (5 pg/50 μl) completely inhibits the development ofdiarrhoea in the IBD-model.

REFERENCES

[0062] 1. Ugwo S. O. et al., J. Pharm. Biomed. Anal. Mar; 19(3-4): p.391-7 (1999);

[0063] 2. Paradossi G. et al., Biopolymers, Aug.; 50(2): p. 201-9(1999).

1. A compound suitable for inhibiting the influx of polymorphonuclearleukocytes (PMNs) into a tissue involved in a chronic inflammatorydisease in a mammal, which compound, in the presence of equimolaramounts of a) N-acetyl-Pro-Gly-Pro (“N-AcPGP”), b) a compound designatedas 4RTR with the formula I

which may form a complex with N-AcPGP, and c) the compound, inhibitscomplex formation between N-AcPGP and 4RTR by at least 10% due toN-AcPGP being bound by the compound.
 2. A compound according to claim 1,which compound produces at least a 25% inhibition of complex formation,preferably at least 50%, more preferably at least 70%, and mostpreferably at least 90%.
 3. A compound according to claim 1 or 2,characterized in that the compound is a peptide.
 4. A compound accordingto claim 1 or 2, characterized in that the compound is a peptidomimetic.5. A method of screening a number of compounds with respect to theircapability to inhibit the formation of a complex between 4RTR andN-AcPGP, screening occurring by means of an assay comprising acompetition reaction between i) a compound A to be assayed and ii) acompound B capable of binding N-AcPGP having an equilibrium constant Kof 5.10⁻⁵ M⁻¹ or lower, compound A and compound B being in competitionfor the binding to a Pro-Gly-Pro (PGP)-comprising compound C, with whichthe compound B is able to complex, having an equilibrium constant K of5.10⁻⁵ M⁻¹ or lower, and wherein a thus assayed compound A is selectedhaving an equilibrium constant K for the reaction between compound A andthe PGP-comprising compound C of 10⁻⁴ M⁻¹ or lower.
 6. A methodaccording to claim 5, characterized in that a compound A is selectedhaving an equilibrium constant K of 10⁻⁵ M⁻¹ or below, preferably 10⁻⁶M⁻¹ or below, more preferably 10⁻⁷ M⁻¹ or below, and most preferably10⁻⁸ M⁻¹ or below.
 7. A method according to claim 5 or 6, characterizedin that the compound C is N-R-Pro-Gly-Pro (“N-R-PGP”), wherein R isbranched or linear alkyl- or acyl group having 1 to 6 carbon atoms.
 8. Amethod according to one of the claims 5 to 7, characterized in that theassay is a homogenous assay.
 9. A method according to one of the claims5 to 8, characterized in that the assay is based on fluorescence(de)polarization or internal energy transfer.
 10. A compound suitablefor inhibiting the influx of PMNs in a tissue involved in a chronicinflammatory disease in a mammal, which compound E, in the presence ofan equimolar amount of N-acetyl-Pro-Gly-Pro (“N-AcPGP”), competes forbinding to a PMN and inhibits the binding of N-AcPGP to the PMN by atleast 10% as a result of N-AcPGP being bound by the compound, and whichcompound E does not induce activation of the PMN.
 11. A compoundaccording to claim 10, which compound E inhibits binding by at least25%, preferably by at least 50%, more preferably by at least 70% andmost preferably by at least 90%.
 12. A compound according to claim 10 or11, characterized in that the compound is a peptide.
 13. A compoundaccording to claim 10 or 11, characterized in that the compound is apeptidomimetic.
 14. A compound according to claim 10 or 11,characterized in that the compound is an antibody, preferably amonoclonal antibody, and most preferably a human or humanized monoclonalantibody, or fragments thereof.
 15. A method of screening a number ofcompounds with respect to their ability to inhibit the binding ofN-AcPGP to a PMN, wherein screening occurs a) by using an assaycomprising a competition reaction between a compound E to be assayed anda compound D for the binding to a PMN, wherein compound D is capable ofcompeting with N-acetyl-Pro-Gly-Pro (“N-AcPGP”) for binding to a PMN andat equimolar concentrations of the compound D and N-AcPGP, inhibits thebinding of N-AcPGP by at least 50%, and at equimolar concentrations ofthe compound E and compound D, inhibits the binding of D to the PMN byat least 10%, and b) by contacting the compound E with a PMN andselecting a compound E which substantially does not induce activation ofthe PMN.
 16. A method according to claim 15, characterized in thatcompound D is N-R-Pro-Gly-Pro, wherein R is a branched or linear alkylor acyl group having 1 to 6 carbon atoms, preferably 1 to 2 carbonatoms, such as N-Ac-PGP or N-methyl-PGP.
 17. A method according to claim15 or 16, characterized in that the assay in respect to the activationof the PMN comprises measuring the polarization of the PMN.
 18. A methodaccording to claim 15 or 16, characterized in that the inhibition of thebinding is established with the aid of flow cytometry.
 19. Anpharmaceutical composition comprising a compound according to one of theclaims 1 to 4 or 10 to 14, or a compound selected with a methodaccording to one of the claims 5 to 9 or 15 to 18, together with apharmaceutically acceptable carrier or excipient.
 20. An application ofa compound according to one of the claims 1 to 4 or 10 to 14, or acompound selected with a method according to one of the claims 5 to 9 or15 to 18 for the preparation of the pharmaceutical composition suitablefor the treatment of a chronic inflammatory disease in a mammal.
 21. Anapplication according to claim 20, characterized in that the chronicinflammatory disease is a disease belonging to the group comprisingchronic inflammatory bowel diseases, rheumatoid arthritis, and otherauto-immune diseases, heart diseases that are characterized by an influxof neutrophilic granulocytes, such as heart ischemia, Adult RespiratoryDistress Syndrome (ARDS), asthma and lung emphysema.